CN101134768A - Method for refining cucurbitacin raw material and preparation thereof - Google Patents

Abstract

The present invention belongs to the field of medicine technology, and provides cucurbitacin refining process and preparation. The cucurbitacin refining process includes the following steps: 1. dissolving marketed cucurbitacin in C1-C4 alcohol-water dissolved ketone to obtain solution A containing cucurbitacin in 0.1-10 g each 100ml; 2. adding active carbon in0.01-10 g for each 100 ml, stirring for 1-60 min and filtering out carbon to obtain solution B; 3. adding water into solution B to reach alcohol/water volume ratio of 1 to 1-10, and stirring until the solution becomes turbid; 4. letting stand for over night; and 5. filtering, collecting precipitate and drying. The refining process can lower the toxicity of cucurbitacin product, has no altering on cucurbitacin B content and is suitable for industrial production. The cucurbitacin product may be prepared into different preparations for treating tumors, increasing peripheral blood leukocytes and treating hepatitis.

Description

A kind of process for purification of cucurbitacin raw material and preparation thereof

Technical field:

The invention belongs to medical technical field, relate to the process for purification of known substance cucurbitacin (Cucurbitacins), and make with extra care the various preparations of feedstock production with this method gained.

Background technology:

Cucurbitacin (Cucurbitacins) belongs to the 19-methyl and appears at a class tetracyclic triterpenoids compound on the C-9 position, mainly be distributed in the cucurbitaceous plant, in high plants such as Cruciferae, scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datiscaceceae and some macro fungis, discovery arranged also.The researcher Qiu Minghua of Kunming plant institute of the Chinese Academy of Sciences leads scientific and technical personnel on the basis of a series of novel cucurbitacins of finding, 229 nearly all cucurbitacine compounds have been studied, new textural classification pattern has been proposed, Qian 5 class formation types are divided into 12 classes again thus, have obtained the approval of international academic community.At present, the cucurbitacin that China's approval is produced is Cucurbitacine again, be Chinese medicine Muskmelon Base (another name: Pedicellus Melo) extract, mainly contain compositions such as Cucurbitacin B, E, Cucurbitacin B content is more than 60% in the existing extract, and the quality standard of cucurbitacin raw material and tablet thereof is recorded in China's ministerial standard.The cucurbitacin sheet is used for the treatment of chronic hepatitis, and (1986,21 (6): 357), through Shanghai, ground 13 tame hospital clinicals such as Beijing, Chongqing, it is efficient to be 75.2%, and obvious effective rate is 44.6% for cucurbitacin sheet, pharmacy circular.Observe the cucurbitacin sheet clinically and can improve chronic hepatitis common sympton and main physical signs more all sidedly, and have and significantly fall enzyme (S-GPT), fall turbid (TTT, ZnTT) and fall the red matter effect of courage, do not cause the S-GPT knock-on after the drug withdrawal, albumen inversion and hyperglobulinemia also there is tangible role of correcting, can also improve the non-specific cell immunological competence of chronic hepatitis patient, no obvious toxic-side effects.With the exception of this, the cucurbitacin sheet can also be used for the treatment of primary hepatocarcinoma, and is efficient 69% through Shanghai, ground such as Beijing, Guangxi six tame hospital clinicals observe, and add up 169 examples, obvious effective rate 39%.Clinical observation shows, this medicine and 5-fluor-uracil are relatively, it improves symptom, eliminate hepatodynia, dwindle the knurl body, prolong lifetime, regain one's strength etc., all be better than control group, and the toxic side effect of chemotherapeutics as none (treatment hepatitis, liver cancer new drug cucurbitacin sheet, herbal medicine 1987,18 (10): 21; The 50 routine clinical observations of cucurbitacin treatment primary hepatocarcinoma, new drug and clinical 1984,3 (2): 21-22; The pharmacology of cucurbitacin and clinical application, herbal medicine, 1992,23 (11): 605～608).The author is arranged with cucurbitacin sheet and chemotherapeutic associating, the treatment mid and late liver cancer, median survival interval was by 6.1 ± 7.12 months of single chemotherapeutic, (the cucurbitacin sheet adds the clinical observation on the therapeutic effect of chemotherapeutic treatment advanced primary liver cancer to bring up to 12.5 ± 7.54 months, cancer, 1989,8 (6): 434-436); The researchist of the U.S. reports recently, and the anticancer mechanism of action of cucurbitacin is inhibition STAT3 activity.Bolleddula Jayaprakasam etc. has compared Cucurbitacin B, D, E, I and the Zorubicin antitumour activity to people's rectum cancer (HCT-116), mammary cancer (MCF-7), lung cancer (NC I-H460) and central nervous system cancer (SF-268) cell, the result shows, Cucurbitacin B when 0.4 μ M, to the inhibiting rate of people's rectum cancer be 81.5%, human breast carcinoma 87%; Inhibiting rate to people's lung cancer when 0.1 μ M is 96%; 0.05 μ M is 92% to the inhibiting rate of people CNS cancer, the inhibiting rate that all is higher than Zorubicin (is respectively 64%, 47%, 45%, 71%, 3 μ M), be that Cucurbitacin B is 7.5,30,60 times of (Bolleddula Jayaprakasam et al.Anticancer and antiinflammatoru activity ofcucurbitacins fromCucurbita andreanaCancer Letter s2003,189:11 of Zorubicin at least to corresponding oncocyte toxicity ^-16).In same piece of writing document, the author finds that also Cucurbitacin B is to the selective restraining effect of COX-2.In addition, the IC of the report anti-liver cancer cell of Cucurbitacin B such as Jud itBartalis (HepG2) ₅₀Be 27.7 μ M, Cucurbitacin E is 15.3 μ M (Judit Bartalis et al Relationsip betweencucurbitacins reversed-phase high-performance liquidchroma tography hydrophobicity index and basal cytotoxicity on HepG2cells Journal of Chromatography B, 818 (2005) 159-166).

Seen that application and disclosed patent have: " nano medicine ' Hulusu ' and preparation method thereof; application (patent) number: 01103658.3 ", " cucurbitacin cyclodextrin inclusion compound and preparation thereof; application (patent) number: 02153647.3 ", " cucurbitacin liposome prescription and preparation thereof; application (patent) number: 02144633.4 ", " drop pills of cucurbitacine and preparation method thereof; application (patent) number: 200310100943.3 ", " drop pills of cucurbitacine and preparation technology thereof; the patent No.: 200310113362.3 ", " cucurbitacin medicinal dropping ball and preparation method thereof; the patent No.: 03134669.3 ", " cucurbitacin liquid type prescription and preparation thereof; the patent No.: 200410021536.8 ", " prescription of drop pills of cucurbitacine preparation and preparation method thereof; the patent No.: ZL200410065085.8 ", " cucurbitacin soft capsule and preparation technology thereof; the patent No.: 200510063660.5 ", " Chinese medicine cucurbitacine soft capsule and preparation method thereof; the patent No.: 200510097038.6 ", " a kind of drop pills of cucurbitacine and preparation method thereof, the patent No.: 200510013633.7 ".

Though so a large amount of patent/document is arranged, there is no research report cucurbitacin process for purification relation toxic with it.The traditional extraction process of cucurbitacin only is to extract after drying, extract obtainedly often presents certain color, in existing literature, does not see the technology that adopts activated carbon treatment, does not also see the report of using the refining various preparations of feedstock production.

Summary of the invention:

Related in the present invention cucurbitacin is by a class tetracyclic triterpenoids compound of extracting in cucurbitaceous plant, Cruciferae, scrophulariaceae, Begoniaceae, Elaeocarpaceae, Datiscaceceae plant and some macro fungis; More particularly, cucurbitacin is the carpopodium (base of fruit of cucurbitaceous plant muskmelon (Cucumismelo L.), muskmelon pedicel) extracts the refining cucurbitacin that obtains (the 19 institute of Drug Standard of Ministry of Public Health of the Peoples Republic of China Chinese traditional patent formulation preparation records), main component is a Cucurbitacin B in the cucurbitacin, Cucurbitacin B content still can comprise multiple compositions such as Cucurbitacin E greater than 60%.

Existing cucurbitacin raw material outward appearance look Huang in order to change the color of raw material, improves the quality of product, and we are surprised to find when it is furtherd investigate, if index composition Cucurbitacin B content is improved, the gained raw material also increases the toxicity of mouse.

Gac is a kind of good sorbent material, and it has very strong adsorptive power to impurity such as pigment, bacterium, pyrogens, and filtrating aid function is arranged.The conventional formulation production technique, as adopting 0.1% (W/V) activated carbon treatment in the injection production technique is in order to remove pyrogen, our result of study shows, with commercially available cucurbitacin raw material with activated carbon treatment after, improved the outward appearance of medicine greatly, obtain the white raw material of outer appearnce to white, dissolution rate improves, what is more important is found obviously to reduce through the toxicity that this step is handled the gained raw material, can increase the antitumous effect of medicine by improving dosage.Simultaneously, we also find, adopt the Cucurbitacin B high-content raw material of the refining marketable material gained of silica gel column chromatography, and as being index with the Cucurbitacin B, content is respectively 80 ^-85%, 85% ^-90%, 90 ^-95% (adopts post to separate, petrol ether/ethyl acetate is a moving phase, after the drying, use ethyl alcohol recrystallization at last, obtain different purity, also guarantee consistent with the raw material residual ethanol of absorbent charcoal fine purification) refining raw material, its toxicity increases with purity, promptly toxicity increases along with the increase of Cucurbitacin B content.Therefore we select the more method of simple possible, promptly use activated carbon treatment, and refining commercially available cucurbitacin raw material particularly cooperates the water of suitable proportion and adds the speed of entry, and the refining raw materials quality of gained is better.

Process for purification of the present invention, can carry out according to following steps:

A, with commercially available cucurbitacin raw material C1 ^-The alcohol of C4 and the letones dissolving miscible with water, making its concentration is 0.1% ^-10%, get A liquid;

B, in A liquid, add 0.01% ^-10% gac stirs appropriate time, filters carbon removal, gets B liquid;

C, be 1 with the volume ratio of water: 1-1 according to alcohol: 10, in B liquid, add entry, stir, to the solution becomes muddiness;

D, stop to stir standing over night;

E, filtration, collecting precipitation, drying, promptly.

Wherein said C ₁ ^-C ₄Alcohol comprises methyl alcohol, ethanol, propyl alcohol (comprising n-propyl alcohol, Virahol), butanols (comprising propyl carbinol, isopropylcarbinol, the trimethyl carbinol); The ketone miscible with water is acetone.Resulting A or B liquid can directly carry out freeze-drying or spraying drying; (when carrying out lyophilize, used best solvent is the trimethyl carbinol).Carrying out pure water when mixing, the ratio control of alcohol and water was at 1: 1 ^-More satisfactory in 1: 10 scope.The raw material of making gained by pure crystal can carry out drying or in room temperature-80 ℃ vacuum-drying in room temperature.After optimizing, best process for refining is as follows:

Get commercially available cucurbitacin raw material 50.0g, its (40-50 ℃ of water-bath) is dissolved among the 95% medicinal alcohol 1500ml, add 3g gac (0.2%), stirring at room 20 minutes is filtered.Add 5000ml water according to the speed that is not more than 1000ml/ minute in the filtrate, opalescence occurs, occur precipitation then, seal, room temperature is placed and is spent the night, and generates a large amount of white precipitates, suction filtration; The gained solid got white refining raw material in 50 ℃ of vacuum-drying 6-12 hours.

Owing to ignored the problems such as color and luster that leaching process may bring in the existing cucurbitacin raw material production technique, thereby it is few for a long time when making in the raw material other irrelevant impurity, shallow when dark during color, these all can cause toxic discordance, the result of treatment of medicine is inconsistent, thereby influence clinical application, it is unfavorable to bring to the patient.Through after the activated carbon treatment, problems such as the color and luster that adopts process for refining of the present invention can get rid of the subtle change on original cucurbitacin production technique may to cause, toxicity variation make the curative effect of Chinese medicine, toxicity data stable, are beneficial to and enter the world market.

Refining cucurbitacin raw material can combine with pharmaceutically acceptable carrier makes oral, injection, external preparation.Preparation comprises tablet, capsule, pill, liposome, nanoparticle, micella, suspensoid, emulsion, solution, ointment, aerosol, nasal drop, nasal spray, suppository, patch etc.The specification of its preparation is 0.1mg-1mg, and human dosage is 0.3mg-3mg/ day, divides 1-4 administration, and independent or drug combination is used for the treatment of various tumours, improves peripheral blood leucocyte, treats hepatitis.

Compare with existing commercially available cucurbitacin raw material, the resulting refining raw material of the present invention has following advantage:

1, the color and luster of refining raw material presents off-white color or white;

2, dissolution rate improves greatly, the raw material that obtains by spraying drying or lyophilize particularly, and the dissolution rate of the refining raw material of gained improves several times than the dissolution rate of original marketable material;

3, toxicity obviously reduces, thereby can improve curative effect by improving dosage;

4, because the composition of the improvement of color and luster, refining raw material changes minimizing, guaranteed the curative effect of made preparation.

Embodiment:

Raw material: cucurbitacin (purchase) in Tianjin Medicine Research Academy Pharmaceutical Co., Ltd; Medium chain triglyceride (MCT buys in Shanghai Dong Shang Industrial Co., Ltd. or Tieling Beiya Medical Oil Co., Ltd.).

Reagent: ethanol (pharmaceutical grade); Methyl alcohol, acetonitrile are chromatographically pure, and other reagent such as propyl alcohol, butanols etc. are analytical pure.

HPLC condition: chromatographic column: Diamonsil C18 (200 * 4.6mm, 5 μ m, Di Ma company); Moving phase: acetonitrile-water-phosphoric acid (45: 55: 0.1); Ultraviolet detection wavelength: 228nm; Column temperature: 30 ℃; Flow velocity: 1mLmin-1; Sample size: 20 μ L.

Animal: all animal-origins are animal housing of Shenyang Pharmaceutical University or animal housing of Chinese Medical Sciences University.

The present invention is provided by the following example, but protection scope of the present invention not only is confined to this.The investigation of embodiment 1 different material consumption

Take by weighing commercially available cucurbitacin raw material 1 gram, 2 grams, 3 grams respectively, use the 60ml dissolve with ethanol respectively, get drug solution.Activated carbon dosage according to 0.1% (W/V) adds gac in the medicine alcoholic solution, and stirring at room was filtered carbon removal after 20 minutes, got solution.Add distilled water 100ml respectively, stirring and evenly mixing, sealing is placed on refrigerator (4 ^-8 ℃) in.Placement is spent the night, and obtains a large amount of white precipitates, the leaching precipitation.Observe outward appearance and weigh calculated yield.The results are shown in Table 1.

The yield result of table 1. different material consumption

Drug weight	1 gram	2 grams	3 grams
				Outward appearance	White	White	Off-white color
Yield (%)	82	86	90

Hence one can see that, and the raw material consumption is big more, and yield is high more.Tracing it to its cause is the influence that the dissolved substance amount accounts for the dosage scale in the final solution, and the raw material consumption is big, and to account for the dosage ratio little for the dissolved substance amount in the final solution, so the yield height.

Embodiment 2 investigates and contains the influence of alcohol amount to yield in the final solution

Take by weighing three parts of commercially available cucurbitacin raw material 2 grams respectively, use 60,80 respectively, the 100ml dissolve with ethanol, drug solution.Activated carbon dosage according to 0.1% (W/V) adds gac in the medicine alcoholic solution, and stirring at room was filtered carbon removal after 20 minutes, got solution.Add distilled water 200ml respectively, stirring and evenly mixing, sealing is placed on refrigerator (4 ^-8 ℃) in.Placement is spent the night, and obtains a large amount of white precipitates, the leaching precipitation.Observe outward appearance and weigh calculated yield.The results are shown in Table 2.

Contain of the influence of alcohol amount in table 2. final solution to yield

Drug weight	2 grams	2 grams	2 grams
				Precipitation outward appearance alcohol adding amount	White 60ml	White 80ml	Off-white color 100ml
Yield (%)	90	86	78

Hence one can see that, and alcohol adding amount is big more, and yield is low more.Trace it to its cause is to contain the alcohol amount in the final solution.Take all factors into consideration, the proportioning of the corresponding 60ml ethanol of 2 gram medicines, 200ml distilled water is adopted in suggestion, i.e. the proportioning of the corresponding 30ml ethanol of 1 gram medicine, 100ml distilled water.

Embodiment 3 investigates the influence of dwell temperature to yield

Take by weighing 3 parts of commercially available cucurbitacin 1 grams respectively, add 30ml ethanol, in 40 ℃ ^-Dissolve in 50 ℃ of water-baths, add 0.1% gac, 40 ℃ were stirred 20 minutes, and added entry 100ml, in 6 ℃ (4 ℃ ^-8 ℃) ,-20 ℃ with room temperature (20 ^-25 ℃) place and spend the night, investigate the sedimentary situation of differing temps, the results are shown in Table 3.

The yield of medicine under table 3. differing temps

	-20℃	6℃	Room temperature
				Yield (%)	90	89	86

Hence one can see that, and the yield of-20 ℃ of following resultant medicines is the highest, but three's difference is not very big.Because-20 ℃ of inconvenient operation, and time-consuming, the cost height is taken all factors into consideration, and can select 6 ℃ or room temperature.

Embodiment 4 adds the influence of water speed to gained crystallization dissolution rate

Get three parts of commercially available cucurbitacin raw material 5.0g, respectively its (40-50 ℃ of water-bath) is dissolved among the 95% medicinal alcohol 150ml, add 0.3g gac (0.2%), stirring at room 20 minutes is filtered respectively, gets three parts of filtrates.Slowly add water 500ml according to the speed of toppling over (about 5000ml/min), about 1000ml/ minute, about 500ml/ minute along wall respectively, opalescence occurs, occur precipitation then, seal, room temperature is placed and is spent the night.Generate a large amount of white precipitates, suction filtration gets white solid.50 ℃ of vacuum-dryings, the gained white solid is about 4.3g, 4.2g, 4.3g respectively.HPLC measures refining cucurbitacin, and Cucurbitacin B content all near 62 μ g/mg, does not have difference with refining preceding marketable material.These three kinds of refining raw materials are used MCT (medium chain triglyceride) dissolving respectively, investigate water and add the influence of speed the dissolution rate of gained highly finished product in MCT, first kind the dissolution rate of toppling over the refining raw material of mode gained is the slowest as a result, then the dissolution rate of two kinds of method gained cucurbitacin highly finished product is obviously accelerated, and both dissolution raties do not have significant difference, therefore, the speed control that we determine to add entry at last promptly adds water speed and is not more than 1000ml/ minute below 1000ml/ minute.

Embodiment 5 vacuum-drying temperature

Take by weighing 3 parts of commercially available cucurbitacin 10 grams respectively, add 300ml ethanol, dissolve in 50 ℃ of water-baths, add 0.1% gac, 50 ℃ were stirred 20 minutes, filtered respectively, got three parts of filtrates.Filtrate is according to adding the speed adding 1000ml water that water speed is not more than 1000ml/ minute, room temperature (20 ^-25 ℃) place and spend the night, filter collecting precipitation, investigate differing temps vacuum-drying to the needed time situation of constant weight, the results are shown in Table 4.

Vacuum-drying is to the test-results of required time of constant weight under table 4. temperature

Temperature (℃)	Room temperature	40	50	60	70	80
							Outward appearance	White	White	White	White	White	White
Time (hour)	12	10	8	6	6	5

The investigation of embodiment 6 different activities charcoal consumptions

Carry out with reference to " embodiment 3 ".Take by weighing several parts of commercially available cucurbitacins, add ethanol respectively, after the dissolving, add 0.01% ^-The gac of 10% (W/V) stirred 20 minutes, filtered respectively.Add entry in the filtrate, room temperature is placed and is spent the night, and obtains a large amount of white precipitates, the leaching precipitation.Observe outward appearance and weigh calculated yield.The results are shown in Table 5.

Table 5. different activities charcoal consumption is to the influence of yield

Activated carbon dosage (%)	0.01	0.05	0.5	1	5	10
							The precipitation outward appearance	The off-white color crust	White	White	White	White	White
Yield (%)	90	91	89	86	85	73

Along with the increase of activated carbon dosage, the yield of medicine descends to some extent, but surpasses at 5% o'clock, and yield obviously reduces, and suggestion is adopted the gac below 5% to carry out medicine in the future and made with extra care.

Embodiment 7 technologies are amplified

Get two parts of commercially available cucurbitacin raw material 50.0g, respectively its (40-50 ℃ of water-bath) is dissolved among the 95% medicinal alcohol 1500ml, add 3g gac (0.2%), stirring at room 20 minutes is filtered.Filtrate slowly adds water 5000ml ml (add water speed and be not more than 1000ml/ minute) along wall, opalescence occurs, occurs precipitation then, seals, and investigates 6 ℃ or room temperature placement and spends the night, and investigates the yield of two kinds of method gained highly finished product.The result generates a large amount of white precipitates, and suction filtration, solid get white solid and are respectively 44.6g, 43.6g in 50 ℃ of vacuum-dryings.Yield is respectively 89.2%, 87.2%.Do not have the significance difference, take all factors into consideration, select room temperature to obtain sedimentary technology.The gained material carries out HPLC to be measured, and Cucurbitacin B content all is about 62 μ g/mg, and Cucurbitacin B content also is about 62 μ g/mg in the marketable material, and main component is not lost, and the peak type on the color atlas, peak number are all with refining preceding consistent.

Embodiment 8 spraying dryings

Take by weighing cucurbitacin raw material 50 grams, use the 2000ml dissolve with ethanol, get drug solution.Add 3 gram gacs according to 0.1% (W/V), stirring at room was filtered carbon removal after 20 minutes, got solution.Carry out spraying drying (70 ℃ of inlet temperatures ^-160 ℃, best 80 ℃ ^-120 ℃; 30 ℃ of temperature outs ^-80 ℃, best 40 ℃ ^-60 ℃; Per minute 5 ^-30ml, best 10ml ^-20ml).Yield is greater than 80% (the part cucurbitacin loses because of sticking wall).

The preparation of embodiment 9 injection rank cucurbitacin raw materials (existing raw material is oral level, no injection stage)

Carry out with reference to " embodiment 3 ".Take by weighing 3 parts of cucurbitacins, add ethanol respectively, after the dissolving, add 0.5% gac, stirred 20 minutes, carry out the essence filter with 0.22 μ m millipore filtration after the coarse filtration, remove charcoal, add sterilized water for injection (perhaps water for injection) in the gained filtrate, room temperature is placed and is spent the night, filter collecting precipitation, drying gets white solid.Check pyrogen or intracellular toxin, 3 batches all qualified, can be used for the preparation of injection.

Execute the test of example 10 dissolution raties

Take by weighing refining raw material 0.01 gram of marketable material and " embodiment 7, embodiment 8 " respectively, add 10 gram MCT, place shaking table, carry out the dissolution rate test under 50 ℃.Triplicate all dissolves the needed time to the results are shown in Table 6.

Whole needed times of material dissolution of table 6. (minute)

Marketable material	18	21	21
				Embodiment 7	12	10	10
Embodiment 8	10	9	11

The result shows that marketable material needs about 20 minutes, and refining raw material only needs about 10 minutes, and the dissolution rate of refining raw material is obviously faster than marketable material.

Embodiment 11 other solvents

According to the method for " embodiment 7 ", as the solvent made from extra care raw material, can obtain same result with methyl alcohol, propyl alcohol (comprising n-propyl alcohol, Virahol), butanols (comprising propyl carbinol, isopropylcarbinol), acetone.

Embodiment 12 freeze-dryings

Get raw material, 40 ℃, with trimethyl carbinol dissolving, add gac, adsorbed 20 minutes, to filter, gained filtrate is carried out lyophilize, gets the white powder solid.This solid carries out the dissolution rate test according to " embodiment 10 ", approximately only needs all to dissolve in 6 minutes, and dissolution rate improves greatly.Embodiment 13 stable form liquid formulas

Best prescription is: 1 part of refining cucurbitacin, 1000 parts of medium chain triglycerides (MCT), 00 part of vitamin e1.

Can prepare soft capsule, liquid-type hard capsule, prepared soft capsule, hard capsule can be oral also can rectum or vagina administration, certainly, liquid itself can direct packaging be used to be coated with in bottle puts administration on the skin; Also can be prepared into nasal drop etc.; In prescription, add emulsifying agent, can be prepared into emulsion.

The accelerated stability and the study on the stability result that keeps sample show, are index with the Cucurbitacin B, adopt this stable form prescription prepared preparation, and the room temperature shelf-stability was at least 2 years.

Embodiment 14 acute toxicity test in mice

The preparation of preparation: according to following prescription preparation marketable material and the refining raw material emulsion of " embodiment 7 " gained.

Cucurbitacin 5g

MCT_ 1000g

Vitamin-E 50g

Phosphatidase 11 60g

HS15 100g

Glycerine 200g

Water adds to 10000ml

10 every group of mouse, male and female half and half.1 gastric infusion, the animal ad lib drinking-water after the administration places the environment of 18-22 to observe 7d.Write down animal toxicity symptom and death toll every day.Dead animal is performed an autopsy on sb, and the situation of visual inspection main organs at last according to institute's administration agent amount and mortality ratio, is calculated LD50 with the Bliss method.The LD50 of marketable material is 8.2mg/kg as a result, and the LD50 of " embodiment 7 " refining raw material is 11.6mg/kg; Intravenous injection, LD50 is 0.91mg/kg, and the LD50 of " embodiment 7 " refining raw material is 1.36mg/kg.Adopt the toxicity of the refining raw material of this patent significantly to reduce.

Embodiment 15 antitumor tests

(1) foundation of bearing mouse model

External recovery liver cancer H22 knurl strain cell, at the collecting cell exponential phase of growth of cell, 1000r/min is centrifugal, PBS washing 2 times, the centrifugal supernatant that goes with the stroke-physiological saline solution dilution, adjusts to 2 * 10 ⁷/ ml.Choose healthy mice, every the above-mentioned cell suspension 0.2ml of usefulness abdominal injection, note observing the ascites growing state of inoculation mouse, about week back inoculation mouse web portion obviously rises greatly, protrudes, extract ascites, ascites is creamy white, and counting concentration with stroke-physiological saline solution dilution oncocyte in sterile test tube is 1 * 10 ⁷Individual/ml, every subcutaneous vaccination 0.2ml sets up the right armpit subcutaneous vaccination of rat liver cancer H22 model.

(2) experiment grouping and administration

With the mouse random packet of modeling success, 10 every group (20 of control groups), numbering in the group.Oral administration route is respectively model control group (blank emulsion); (emulsion is irritated stomach to commercially available cucurbitacin raw material group, and dosage is according to total cucurbitacin meter, 0.15mgkg ^-1); Refining raw material group (is irritated stomach dosage according to total cucurbitacin meter, 0.20mgkg ^-1); The intravenous administration approach is respectively model control group (blank emulsion); (dosage is according to total cucurbitacin meter, 0.08mgkg for emulsion, intravenous injection for commercially available cucurbitacin raw material group ^-1); (intravenous injection, dosage is according to total cucurbitacin meter, 0.12mgkg for refining raw material group ^-1).Beginning administration in second day after modeling, successive administration 10 days, once a day.

(3) claim that knurl is heavy and calculate tumour inhibiting rate

Drug withdrawal 24h, the cervical vertebra dislocation method is put to death animal, weighs, and dissects and peels off the knurl piece, and electronic balance claims knurl heavy.Calculate tumour inhibiting rate according to following formula:

Tumour inhibiting rate=(the average knurl of the average knurl weight/control group of 1-administration group is heavy) * 100%

As a result, oral route, the tumour inhibiting rate of commercially available cucurbitacin raw material group and refining raw material group is respectively 30.3%, 43.1%; The intravenous injection approach, the tumour inhibiting rate of commercially available cucurbitacin raw material group and refining raw material group is respectively 37.2%, 53.8%.And the peripheral blood leucocyte of mouse is consistent with the white corpuscle of normal blank group, does not have significant difference.In one group of other experiment, find, with the coupling of chemotherapeutics 5 FU 5 fluorouracil, can the raise numerical value of the leukopenia that damage because of 5 FU 5 fluorouracil causes of the refining cucurbitacin of gained of the present invention, 5 FU 5 fluorouracil group mouse peripheral blood leucocyte descends very significantly, and logotype group mouse peripheral blood leucocyte does not significantly reduce, and is consistent with normal blank group.

Obviously, adopt the refining raw material of gained of the present invention, can increase the antitumous effect of medicine, also have the effect that the protection chemotherapeutics causes leukopenia by improving dosage.

The comparison of embodiment 16 marketable material and refining raw material

The refining raw material of marketable material with " embodiment 7 " gained compared, and index is respectively color, the dissolution rate in MCT, injected in mice route of administration acute toxicity, anti-H22 tumor promotion.

The results are shown in Table 7.

The comparison of table 7. marketable material and refining raw material

	Marketable material	" embodiment 7 " make with extra care raw material
			The dissolution rate injected in mice route of administration acute toxicity LD50 of color in MCT	Faint yellow oral slowly: 8.2mg/kg; Injection: 0.91mg/kg	Off-white color-white is oral soon: 11.6mg/kg; Injection: 1.36mg/kg
Anti-H22 tumor promotion	Oral: 30.3%; Injection: 37.2%	Oral: 43.1%; Injection: 53.8%

Embodiment 17 adopts the refining various preparations of feedstock production

Adopt the made raw material of technology of the present invention, can prepare various preparations, as tablet hard capsule soft capsule dripping pill emulsion liposome the protein nano grain injection nasal drop ointment patch vaginal preparation rectal formulation etc.Its clinical using dosage is constant or can suitably improve according to the acute toxicity test in mice data.

Tablet formulation (1000, the 0.1mg/ sheet)

Cucurbitacin 100mg

Lactose 100g

Cross-linked pvp 10g

Micropowder silica gel 1g

Stearic acid 0.5g

Preparation technology: take by weighing the recipe quantity cucurbitacin, use suitable dissolve with ethanol, standby; The lactose of recipe quantity, partial cross-linked PVP and part micropowder silica gel are mixed,, cross 40 orders with PVP ethanolic soln system softwood, the preparation wet granular, 50 ℃ of air seasoning 1 hour, blank particle; Add the cucurbitacin ethanolic soln, cross 40 mesh sieves and carry out whole grain 50 ℃ of air seasoning half an hour, add remaining cross-linked pvp and micropowder silica gel, and add stearic acid, behind the mixing, measure intermediate content, compressing tablet, promptly.

Hard capsule (1000, the 0.3mg/ grain)

Cucurbitacin 300mg

Lactose 150g

Vitamins C 1g

Micropowder silica gel 1g

Talcum powder 1g

Preparation technology: adopt and mix comminuting method,, cross 100 mesh sieves, add the micropowder silica gel and the talcum powder of recipe quantity, mix with cucurbitacin and lactose mixing pulverizing; Measure intermediate content, packing, promptly.

Pill (1000, the 0.1mg/ ball)

Cucurbitacin 0.1g

Polyethylene glycol 6000 40g

Poly(oxyethylene glycol) 400 10g

Butylhydroxy anisole 0.01g

Preparation technology: do not use after the poly(oxyethylene glycol) 400 dissolving of the cucurbitacin that takes by weighing recipe quantity with recipe quantity; Take by weighing the polyethylene glycol 6000 and the butylhydroxy anisole of recipe quantity,, add the poly(oxyethylene glycol) 400 solution of cucurbitacin in 90 ℃ of heating and meltings, behind the mixing,, splash in the dimethyl silicone oil according to the speed of 60 of per minutes with the water dropper of diameter 4.2mm, cool off, promptly get the drop pills of cucurbitacine agent.

Other preparation as emulsion, liposome, protein nano grain, injection, nasal drop, ointment (1mg/g), patch (1mg/ subsides), vaginal suppository, rectal suppository, enema etc., gets final product according to the preparation of conventional formulation prescription.

Embodiment 18 tumour patient clinical applications

The patient of clinical new diagnosis, comprise primary hepatocarcinoma, the liver cancer lung shifts, lung cancer, the lung cancer hepatic metastases, the lung cancer lymphatic metastasis, the rectum cancer, mammary cancer, the mammary cancer lymphatic metastasis, carcinoma of the pancreas, cancer of the stomach, people surplus the lymphoma etc. 20, take the soft capsule of " embodiment 13 ", hard capsule or emulsion, dosage is determined according to patient's situation, be 0.3mg-3mg/ day, divide 1-4 administration, all obtain good therapeutic action, particularly in the quality of making the life better, appetite stimulator, reduce tire first sphaeroprotein (AFP), the effect that improves aspects such as white cell is remarkable, the patient who has took 3 years so far, and tumour is controlled fully, and dwindles gradually.

Claims (9)

1. the process for purification of a cucurbitacin raw material is characterized in that carrying out according to the following step: a, with commercially available cucurbitacin raw material C ₁-C ₄Alcohol and with the dissolving of the miscible letones of water, A liquid, drug level is to contain cucurbitacin 0.1～10g among every 100ml;

B, in A liquid, add 0.01～10g gac, stirred 1～60 minute, filter carbon removal according to every 100ml, must B liquid;

C, be 1 with the volume ratio of water: 1-1 according to alcohol: 10, in B liquid, add entry, stir, to the solution becomes muddiness;

D, stop to stir standing over night;

E, filtration, collecting precipitation, drying, promptly.

2. the process for purification of a kind of cucurbitacin raw material according to claim 1 is characterized in that: said C ₁-C ₄Alcohol comprises methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol; The ketone miscible with water is acetone.

3. the process for purification of a kind of cucurbitacin raw material according to claim 1, it is characterized in that: the optimum concentration range of A liquid is 1%-5%g/ml; The gac optimum quantity that is added is 0.2%-2%g/ml.

4. the process for purification of a kind of cucurbitacin raw material according to claim 1, it is characterized in that: said A or B liquid can directly carry out freeze-drying or spraying drying; When carrying out lyophilize, used solvent is the trimethyl carbinol.

5. the process for purification of a kind of cucurbitacin raw material according to claim 1 is characterized in that: carrying out pure water when mixing, alcohol is 1 with the volume ratio optimum value of water: 4-1: 6; The speed that adds entry is not more than 1000ml/ minute; The raw material of making gained by pure crystal carries out drying or vacuum-drying in room temperature.

6. the process for purification of a cucurbitacin raw material is characterized in that: get commercially available cucurbitacin raw material 50.0g, it is dissolved under 40-50 ℃ of water-bath among the 95% medicinal alcohol 1500ml, add the 3g gac, stirring at room 20 minutes, filtration; Add 5000ml water according to the speed that is not more than 1000ml/ minute, opalescence occurs, occur precipitation then, seal, room temperature is placed and is spent the night, and generates a large amount of white precipitates, suction filtration; The gained solid got white refining raw material in 50 ℃ of vacuum-drying 6-12 hours.

7. the preparation of a refining cucurbitacin raw material is characterized in that: claim 1 or 6 described refining cucurbitacin raw materials can combine with pharmaceutically acceptable carrier makes oral, injection, external preparation.

8. the preparation of a kind of refining cucurbitacin raw material according to claim 7, it is characterized in that: described preparation comprises solid, semisolid, liquid preparation, as tablet, capsule, pill, liposome, nanoparticle, micella, suspensoid, emulsion, solution, ointment, aerosol, nasal drop, nasal spray, suppository, patch.

9. the preparation of a kind of refining cucurbitacin raw material according to claim 8, it is characterized in that: the specification of its preparation is 0.1mg-1mg, human dosage is 0.3mg-3mg/ day, divide 1-4 administration, independent or drug combination is used to prepare the medicine for the treatment of various tumours, raising peripheral blood leucocyte, reaching treatment hepatitis.